This application has two main goals: 1) to understand how the novel transcription factor Isx regulates gut differentiation and alpha-defensin genes, and 2) to finalize my training in laboratory science to become an independent physician-scientist, using the preceding goal as the platform. The program will span five years under the mentorship of my co-sponsors Daniel Podolsky and Ramesh Shivdasani, experts from distinct backgrounds who will complement each other in guiding my scientific and career development. The laboratory project focuses on elucidating regulation of enteric alpha-defensin/cryptdin expression in Paneth and other gut epithelial cells. Recent studies suggest that the canonical Wnt pathway activates at least one portion of the Paneth-cell program: expression of many cryptdins. Independently, Nod2, a gene that is implicated in human Crohn's disease pathogenesis, also regulates expression levels of selected alpha- defensins. I recently identified a novel, intestine-restricted, homeodomain-containing transcription factor, Isx. Studies on the Isx-null ileum reveal massively elevated levels of cryptdin 4 and selected cryptdin-related sequences (Defcr-rs's). Accordingly, we hypothesize that Isx plays a pivotal role in regulating (repressing) their expression, possibly by complementing the independent regulation of Paneth cell differentiation by the Wnt pathway. I propose to elucidate how Isx regulates Paneth cell-specific genes (Specific Aim 1) and to understand the functional consequence of abnormally high levels of cryptdins and Defcr-rs peptides in host susceptibility to mucosal infection and inflammation (Specific Aim 2). First, to investigate the role of Isx in alpha-defensin gene regulation, I will use Isx-null and TOP-GAL transgenic (reporter) mice, which express E. coli LacZ under the control of a Wnt-responsive promoter. I also propose to dissect how Isx regulates expression of cryptdins and Defcr-rs peptides by manipulating Isx levels in cultured cells and through analysis of regulatory c/s-elements in mouse cryptdin and Defcr-rs genes. Second, to elucidate the immunologic significance of highly elevated levels of cryptdin 4 and of a select group of cryptdin- related peptides in Isx-null mice, I will challenge these mutants with chemical, bacterial, and genetic models of inflammation and infection. Together, these studies represent novel approaches toward understanding fundamental aspects of intestinal biology that are pertinent to prevalent clinical disorders. Relevance: Over 500,000 people in the U.S. alone are affected by debilitating Crohn's disease, and current management is based mostly on anti-inflammatory drugs. We need to develop new treatment options that will prevent this disease or achieve sustained remission. Given that several recent studies directly link cryptdins to inflammatory bowel disease (IBD), especially Crohn's disease, the proposed research project has significant clinical implications for understanding and developing ways to better manage IBD.